Pyrazolopyrimidine compounds

ABSTRACT

The present teachings provide a compound represented by the following structural formula: (Formula (I)); or a pharmaceutically acceptable salt thereof. Also described are pharmaceutical compositions and methods of use thereof.

RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/CA2013/000957, filed Nov. 15, 2013. The entire teachings of theaforementioned application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Protein kinases have been the subject of extensive study in the searchfor new therapeutic agents in various diseases, for example, cancer.Protein kinases are known to mediate intracellular signal transductionby effecting a phosphoryl transfer from a nucleoside triphosphate to aprotein acceptor that is involved in a signaling pathway. There are anumber of kinases and pathways through which extracellular and otherstimuli cause a variety of cellular responses to occur inside the cell.

Human TTK protein kinase (TTK), also known as tyrosine threonine kinase,dual specificity protein kinase TTK, Monopolar Spindle 1 (Mps1) andPhosphotyrosine-Picked Threonine Kinase (PYT), is a conservedmultispecific kinase that is capable of phosphorylating serine,threonine and tyrosine residues when expressed in E. coli (Mills et al.,J. Biol. Chem. 22(5): 16000-16006 (1992)). TTK mRNA is not expressed inthe majority of physiologically normal tissues in human (Id). TTK mRNAis expressed in some rapidly proliferating tissues, such as testis andthymus, as well as in some tumors (for example, TTK mRNA was notexpressed in renal cell carcinoma, was expressed in 50% of breast cancersamples, was expressed in testicular tumors and ovarian cancer samples)(Id). TTK is expressed in some cancer cell lines and tumors relative tonormal counterparts (Id.; see also WO 02/068444 A1).

Therefore, agents which inhibit a protein kinase, in particular TTK,have the potential to treat cancer. There is a need for additionalagents which can act as protein kinase inhibitors, in particular TTKinhibitors.

In addition, cancer recurrence, drug resistance or metastasis is one ofthe major challenges in cancer therapies. Cancer patients who respondedfavorably to the initial anti-cancer therapy often develop drugresistance and secondary tumors that lead to the relapse of the disease.Recent research evidences suggest that the capability of a tumor to growand propagate is dependent on a small subset of cells within the tumor.These cells are termed tumor-initiating cells (TICs) or cancer stemcells. It is thought that the TICs are responsible for drug resistance,cancer relapse and metastasis. Compounds that can inhibit the growth andsurvival of these tumor-initiating cells can be used to treat cancer,metastasis or prevent recurrence of cancer. Therefore, a need exists fornew compounds that can inhibit the growth and survival oftumor-imitating cells.

SUMMARY OF THE INVENTION

Applicants have now discovered that certain pyrazolopyrimidine compoundsare potent kinase inhibitors, such as TTK protein kinase (see ExampleB). Applicants have also discovered that these compounds have potentanticancer activity against breast cancer, colon cancer, and ovariancancer cells in cell culture study (see Examples C-D). Based on thesediscoveries, pyrazolopyrimidine compounds, pharmaceutical compositionsthereof, and methods of treating cancer with the pyrazolopyrimidinecompounds are disclosed herein.

The present teachings are directed, at least in part, to a compoundrepresented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is NH—CH₂-Cy,    -   Cy is C₃-C₄ cycloalkyl optionally substituted with one or two        groups selected from alkyl and hydroxyl;    -   R² is —O-pyridinyl; —NH—(C₂-C₆)hydroxyalkyl optionally        substituted with cyclopropyl or isopropyl; or        —NH—(C₃-C₆)cycloalkyl optionally substituted with hydroxyl or        (C₁-C₂)hydroxylalkyl;    -   R⁴ is selected from hydrogen, halogen, and (C₁-C₃)alkyl; and        R^(d) is cyclopropyl. Preferably, R⁴ is chlorine or methyl.

In one embodiment, the present teachings include a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand a compound represented by structural formula (I) described above ora pharmaceutically acceptable salt thereof.

In another embodiment, the present teachings provide a method oftreating a subject having cancer comprising administering to the subjectan effective amount of a compound of structural formula (I) or apharmaceutically acceptable salt thereof.

Another embodiment of the present teachings provides a method ofinhibiting TTK activity in a subject in need of inhibition of TTKactivity, comprising administering to the subject an effective amount ofa compound represented by Structural Formula (I) or a pharmaceuticallyacceptable salt thereof.

Another embodiment of the present teachings includes the use of acompound represented by Structural Formula (I) or a pharmaceuticallyacceptable salt thereof in therapy. In some embodiments, the therapy isfor treating a subject with cancer. Alternatively, the therapy is forinhibiting TTK activity in a subject in need of inhibition of TTKactivity.

Another embodiment of the present teachings includes the use of acompound represented by Structural Formula (I) or a pharmaceuticallyacceptable salt thereof for the manufacture of a medicament for treatinga subject with cancer.

Another embodiment of the present teachings includes the use of acompound represented by Structural Formulas (I) or a pharamceuticallyacceptable salt thereof for the manufacture of a medicament forinhibiting TTK activity in a subject in need of inhibition of TTKactivity.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present teachings are directed to a compoundrepresented by Structural Formula (I) or a pharmaceutically acceptablesalt thereof. The invention also includes the compounds depicted bystructure and/or described by name in the Exemplification, and includesboth the neutral forms and as well as pharmaceutically acceptable saltsthereof. Treatments with and/or uses of these compounds (includingneutral forms and pharmaceutically acceptable salts thereof) asdescribed herein are also included in the invention. Specific examplesof compounds of the invention are shown below:

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof;

or a pharmaceutically acceptable salt thereof; and

or a pharmaceutically acceptable salt thereof.

The term “alkyl” used alone or as part of a larger moiety, such as“hydroxyalkyl”, and the like, means saturated aliphatic straight-chainor branched monovalent hydrocarbon radical. Unless otherwise specified,an alkyl group typically has 1-6 carbon atoms, i.e., (C₁-C₆)alkyl. Asused herein, a “(C₁-C₆)alkyl” group is means a radical having from 1 to6 carbon atoms in a linear or branched arrangement.

“Cycloalkyl” means a saturated aliphatic cyclic hydrocarbon radicaloptionally containing one or more double bonds. It can be monocyclic,bicyclic (e.g., a bridged bicyclic ring), polycyclic (e.g., tricyclic),or fused. For example, monocyclic (C₃-C₇)cycloalkyl means a radicalhaving from 3-7 carbon atoms arranged in a monocyclic ring. A(C₃-C₇)cycloalkyl includes, but is not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

Certain of the compounds described herein may exist in variousstereoisomeric or tautomeric forms. Stereoisomers are compounds whichdiffer only in their spatial arrangement. When a disclosed compound isnamed or depicted by structure without indicating stereochemistry, it isunderstood that the name or structure encompasses all possiblestereoisomers, tautomers, geometric isomers or a combination thereof.

When a geometric isomer is depicted by name or structure, it is to beunderstood that the geometric isomeric purity of the named or depictedgeometric isomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure byweight. Geometric isomeric purity is determined by dividing the weightof the named or depicted geometric isomer in the mixture by the totalweight of all of the geomeric isomers in the mixture.

Racemic mixture means 50% of one enantiomer and 50% of is correspondingenantiomer. The present teachings encompass all enantiomerically-pure,enantiomerically-enriched, diastereomerically pure, diastereomericallyenriched, and racemic mixtures, and diastereomeric mixtures of thecompounds described herein.

Enantiomeric and diastereomeric mixtures can be resolved into theircomponent enantiomers or stereoisomers by well known methods, such aschiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers anddiastereomers can also be obtained from diastereomerically- orenantiomerically-pure intermediates, reagents, and catalysts by wellknown asymmetric synthetic methods.

When a compound is designated by a name or structure that indicates asingle enantiomer, unless indicated otherwise, the compound is at least60%, 70%, 80%, 90%, 99% or 99.9% optically pure (also referred to as“enantiomerically pure”). Optical purity is the weight in the mixture ofthe named or depicted enantiomer divided by the total weight in themixture of both enantiomers.

When the stereochemistry of a disclosed compound is named or depicted bystructure, and the named or depicted structure encompasses more than onestereoisomer (e.g., as in a diastereomeric pair), it is to be understoodthat one of the encompassed stereoisomers or any mixture of theencompassed stereoisomers are included. It is to be further understoodthat the stereoisomeric purity of the named or depicted stereoisomers atleast 60%, 70%, 80%, 90%, 99% or 99.9% by weight. The stereoisomericpurity in this case is determined by dividing the total weight in themixture of the stereoisomers encompassed by the name or structure by thetotal weight in the mixture of all of the stereoisomers.

Included in the present teachings are pharmaceutically acceptable saltsof the compounds disclosed herein. The disclosed compounds have basicamine groups and therefore can form pharmaceutically acceptable saltswith pharmaceutically acceptable acid(s). Suitable pharmaceuticallyacceptable acid addition salts of the compounds described herein includesalts of inorganic acids (such as hydrochloric acid, hydrobromic,phosphoric, nitric, and sulfuric acids) and of organic acids (such as,acetic acid, benzenesulfonic, benzoic, methanesulfonic, andp-toluenesulfonic acids). Compounds of the present teachings with acidicgroups such as carboxylic acids can form pharmaceutically acceptablesalts with pharmaceutically acceptable base(s). Suitablepharmaceutically acceptable basic salts include ammonium salts, alkalimetal salts (such as sodium and potassium salts) and alkaline earthmetal salts (such as magnesium and calcium salts). Compounds with aquaternary ammonium group also contain a counteranion such as chloride,bromide, iodide, acetate, perchlorate and the like. Other examples ofsuch salts include hydrochlorides, hydrobromides, sulfates,methanesulfonates, nitrates, benzoates and salts with amino acids suchas glutamic acid.

Compounds described herein can inhibit various kinases, including theTTK. Thus, generally, compounds described herein are useful in thetreatment of diseases or conditions associated with such kinases. Insome embodiments, compounds described herein can inhibit TTK.

In one embodiment, the compounds described herein are TTK inhibitors,and are useful for treating diseases, such as cancer, associated withsuch kinase(s).

Another aspect of the present teachings relates to a method of treatinga subject with cancer comprising administering to the subject aneffective amount of a compound described herein. In one embodiment, thecompounds described herein inhibit the growth of a tumor. For example,the compounds described herein inhibit the growth of a tumor thatoverexpresses TTK.

Cancers that can be treated (including reduction in the likelihood ofrecurrence) by the methods of the present teachings include lung cancer,breast cancer, colon cancer, brain cancer, neuroblastoma, prostatecancer, melanoma, glioblastoma multiform, ovarian cancer, lymphoma,leukemia, melanoma, sarcoma, paraneoplasia, osteosarcoma, germinoma,glioma and mesothelioma. In one embodiment, the cancer is selected fromleukemia, acute myeloid leukemia, chronic myelogenous leukemia, breastcancer, brain cancer, colon cancer, colorectal cancer, head and neckcancer, hepatocellular carcinoma, lung adenocarcinoma, metastaticmelanoma, pancreatic cancer, prostate cancer, ovarian cancer and renalcancer. In one embodiment, the cancer is lung cancer, colon cancer,brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastomamutiform or ovarian cancer. In another embodiment, the cancer ispancreatic cancer, prostate cancer, lung cancer, melanoma, breastcancer, colon cancer, or ovarian cancer. In yet another embodiment, thecancer is breast cancer, colon cancer and ovarian cancer. In yet anotherembodiment, the cancer is a breast cancer. In yet another embodiment,the cancer is a basal sub-type breast cancer or a luminal B sub-typebreast cancer. In yet another embodiment, the cancer is a basal sub-typebreast cancer that overexpresses TTK. In yet another embodiment, thebasal sub-type breast cancer is ER (estrogen receptor), HER2 and PR(progesterone receptor) negative breast cancer. In yet anotherembodiment, the cancer is a soft tissue cancer. A “soft tissue cancer”is an art-recognized term that encompasses tumors derived from any softtissue of the body. Such soft tissue connects, supports, or surroundsvarious structures and organs of the body, including, but not limitedto, smooth muscle, skeletal muscle, tendons, fibrous tissues, fattytissue, blood and lymph vessels, perivascular tissue, nerves,mesenchymal cells and synovial tissues. Thus, soft tissue cancers can beof fat tissue, muscle tissue, nerve tissue, joint tissue, blood vessels,lymph vessels, and fibrous tissues. Soft tissue cancers can be benign ormalignant. Generally, malignant soft tissue cancers are referred to assarcomas, or soft tissue sarcomas. There are many types of soft tissuetumors, including lipoma, lipoblastoma, hibernoma, liposarcoma,leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma,schwannoma (neurilemoma), neuroma, malignant schwannoma,neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovialsarcoma, hemangioma, glomus tumor, hemangiopericytoma,hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma,fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma,fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP),malignant fibrous histiocytoma (MFH), myxoma, granular cell tumor,malignant mesenchymomas, alveolar soft-part sarcoma, epithelioidsarcoma, clear cell sarcoma, and desmoplastic small cell tumor. In aparticular embodiment, the soft tissue cancer is a sarcoma selected fromthe group consisting of a fibrosarcoma, a gastrointestinal sarcoma, aleiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphicliposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, anda synovial sarcoma.

In some embodiments, the present teachings provide methods of inhibitingthe growth of tumor-initiating cells or reducing the likelihood ofrecurrence of a cancer in a subject who is undergoing an anti-cancertherapy. The method comprises the steps of:

a) assessing the subject to determine whether the cancer is inremission; and

b) if the cancer is in remission; then administering to the subject aneffective amount of a TTK inhibitor (e.g., a compound represented byStructural Formula (I). If the cancer is not in remission, the methodoptionally further comprises the step of continuing the anti-cancertherapy until the cancer goes into remission and then the step b) ofadministering an effective amount of a TTK inhitior (e.g., a compoundrepresented by Structural Formula (I).

As used herein, the term “tumor-initiating cells” or “TICs” refer tocells present within some tumors that possess the ability to self-renewand proliferate. These cells are sometimes called cancer stem cells(CSCs) and may be observed to share certain characteristics with normalstem cells, including a stem celllike phenotype and function. In someembodiments, TICs are characterized by their ability to form tumorsafter xenotransplantation in immunodeficient mice.

In some embodiments, the present teachings provide methods of inhibitingthe growth of tumor-initiating cells or reducing the likelihood ofrecurrence of a cancer in a subject whose cancer is in remissioncomprising administering to the subject an effective amount of a TTKinhibitor (e.g, a compound represented by Structural Formula (I)).

In some embodiments, e.g., where the subject is being treated to reducethe likelihood of recurrence of a cancer, the subject has already beentreated with an anti-cancer therapy. Alternatively, the subject hasalready been treated with an anti-cancer therapy and the subject is inremission.

In some embodiments, the present teachings provide methods of treating asubject with a cancer comprising administering to the subject aneffective amount of a compound represented by Structural Formula (I) incombination with an effective anti-cancer therapy. In one embodiment,the cancer is a metastatic cancer. A “metastatic cancer” is a cancerthat has spread from its primary site to other parts of the body.

In another embodiment, the present teachings are directed to a method oftreating a subject with a drug-resistant cancer. A “drug-resistantcancer” is a cancer that is not responsive to one, two, three, four,five or more drugs that are typically used for the treatment of thecancer. In one embodiment, the drug-resistant cancer is mediated by thegrowth of tumor-initiating cells.

Suitable methods known in the art can be used for assessing a subject todetermine whether the cancer is in remission. For example, the size ofthe tumor and/or tumor markers, usually proteins associated with tumors,can be monitored to determine the state of the cancer. Size of the tumorcan be monitored with imaging devices, such as X-ray, MRI, CAT scans,ultrasound, mammography, PET and the like or via biopsy.

For methods described herein, e.g., coadministration methods, theanti-cancer therapy are selected from the group consisting of surgery,radiation therapy, immunotherapy, endocrine therapy, gene therapy andadministration of an anti-cancer agent. Alternatively, the anti-cancertherapy is radiation therapy. In another alternative, the anti-cancertherapy is immunotherapy. In another alternative, the anti-cancertherapy is administration of an anti-cancer agent. In yet anotheralternative, the anti-cancer therapy is surgery.

Radiation therapy is the use of radiation to kill, destroy or treat thecancers. Exemplary radiation therapy includes, but is not limited to,gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy,proton therapy, brachytherapy, and radioiosotope thereapy (i.e.,systemic radioactive isotopes therapy),

An endocrine therapy is a treatment that adds, blocks or removeshormones. For example, chemotherapeutic agents that can block theproduction or activity of estrogen have been used for treating breastcancer. In addition, hormonal stimulation of the immune system has beenused to treat specific cancers, such as renal cell carcinoma andmelanoma. In one embodiment, the endocrine therapy comprisesadministration of natural hormones, synthetic hormones or othersynthetic molecules that may block or increase the production of thebody's natural hormones. In another embodiment, the endocrine therapyincludes removal of a gland that makes a certain hormone.

As use herein, a gene therapy is the insertion of genes into a subject'scell and biological tissues to treat diseases, such as cancer. Exemplarygene therapy includes, but is not limited to, a germ line gene therapyand a somatic gene therapy.

Immunotherapy (also called biological response modifier therapy,biologic therapy, biotherapy, immune therapy, or biological therapy) istreatment that uses parts of the immune system to fight diseaseImmunotherapy can help the immune system recognize cancer cells, orenhance a response against cancer cells. Immunotherapies include activeand passive immunotherapies. Active immunotherapies stimulate the body'sown immune system while passive immunotherapies generally use immunesystem components created outside of the body.

Examples of active immunotherapies include, but are not limited tovaccines including cancer vaccines, tumor cell vaccines (autologous orallogeneic), dendritic cell vaccines, antigen vaccines, anti-idiotypevaccines, DNA vaccines, viral vaccines, or Tumor-Infiltrating Lymphocyte(TIL) Vaccine with Interleukin-2 (IL-2) or Lymphokine-Activated Killer(LAK) Cell Therapy.

Examples of passive immunotherapies include but are not limited tomonoclonal antibodies and targeted therapies containing toxins.Monoclonal antibodies include naked antibodies and conjugated monoclonalantibodies (also called tagged, labeled, or loaded antibodies). Nakedmonoclonal antibodies do not have a drug or radioactive materialattached whereas conjugated monoclonal antibodies are joined to, forexample, a chemotherapy drug (chemolabeled), a radioactive particle(radiolabeled), or a toxin (immunotoxin). Examples of these nakedmonoclonal antibody drugs include, but are not limited to Rituximab(Rituxan), an antibody against the CD20 antigen used to treat, forexample, B cell non-Hodgkin lymphoma; Trastuzumab (Herceptin), anantibody against the HER2 protein used to treat, for example, advancedbreast cancer; Alemtuzumab (Campath), an antibody against the CD52antigen used to treat, for example, B cell chronic lymphocytic leukemia(B-CLL); Cetuximab (Erbitux), an antibody against the EGFR protein used,for example, in combination with irinotecan to treat, for example,advanced colorectal cancer and head and neck cancers; and Bevacizumab(Avastin) which is an antiangiogenesis therapy that works against theVEGF protein and is used, for example, in combination with chemotherapyto treat, for example, metastatic colorectal cancer. Examples of theconjugated monoclonal antibodies include, but are not limited toRadiolabeled antibody Ibritumomab tiuxetan (Zevalin) which deliversradioactivity directly to cancerous B lymphocytes and is used to treat,for example, B cell non-Hodgkin lymphoma; radiolabeled antibodyTositumomab (Bexxar) which is used to treat, for example, certain typesof non-Hodgkin lymphoma; and immunotoxin Gemtuzumab ozogamicin(Mylotarg) which contains calicheamicin and is used to treat, forexample, acute myelogenous leukemia (AML). BL22 is a conjugatedmonoclonal antibody for treating, for example, hairy cell leukemia,immunotoxins for treating, for example, leukemias, lymphomas, and braintumors, and radiolabeled antibodies such as OncoScint for example, forcolorectal and ovarian cancers and ProstaScint for example, for prostatecancers.

Further examples of therapeutic antibodies that can be used include, butare not limited to, HERCEPTIN® (Trastuzumab) (Genentech, CA) which is ahumanized anti-HER2 monoclonal antibody for the treatment of patientswith metastatic breast cancer; REOPRO® (abciximab) (Centocor) which isan anti-glycoprotein Hb/IIIa receptor on the platelets for theprevention of clot formation; ZENAPAX® (daclizumab) (RochePharmaceuticals, Switzerland) which is an immunosuppressive, humanizedanti-CD25 monoclonal antibody for the prevention of acute renalallograft rejection; PANOREX™ which is a murine anti-17-IA cell surfaceantigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics);LYMPHOCIDE™ Y-90 (Immunomedics); Lymphoscan (Tc-99m-labeled;radioimaging; Immunomedics); Nuvion (against CD3; Protein Design Labs);CM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is aprimatied anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is aradiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 isa humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatizedanti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab);CD20-sreptdavidin (+biotin-yttrium 90; NeoRx); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 is a human anti-TGF-β₂ antibody (Cambridge Ab Tech).

Immunotherapies that can be used in the present teachings includeadjuvant immunotherapies. Examples include cytokines, such asgranulocyte-macrophage colony-stimulating factor (GM-CSF),granulocyte-colony stimulating factor (G-CSF), macrophage inflammatoryprotein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6,IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors(including TNF-alpha), and interferons (including IFN-alpha, IFN-beta,and IFN-gamma); aluminum hydroxide (alum); Bacille Calmette-Gurin (BCG);Keyhole limpet hemocyanin (KLH); Incomplete Freund's adjuvant (IFA);QS-21; DETOX; Levamisole; and Dinitrophenyl (DNP), and combinationsthereof, such as, for example, combinations of, interleukins, forexample, IL-2 with other cytokines, such as IFN-alpha.

Alternatively, the anti-cancer therapy described herein includesadministration of an anti-cancer agent. An “anti-cancer agent” is acompound, which when administered in an effective amount to a subjectwith cancer, can achieve, partially or substantially, one or more of thefollowing: arresting the growth, reducing the extent of a cancer (e.g.,reducing size of a tumor), inhibiting the growth rate of a cancer, andameliorating or improving a clinical symptom or indicator associatedwith a cancer (such as tissue or serum components) or increasinglongevity of the subject.

The anti-cancer agent suitable for use in the methods described hereinincludes any anti-cancer agents that have been approved for thetreatment of cancer. In one embodiment, the anti-cancer agent includes,but is not limited to, a targeted antibody, an angiogenesis inhibitor,an alkylating agent, an antimetabolite, a vinca alkaloid, a taxane, apodophyllotoxin, a topoisomerase inhibitor, a hormonal antineoplasticagent and other antineoplastic agents.

Examples of alkylating agents useful in the methods of the presentteachings include but are not limited to, nitrogen mustards (e.g.,mechloroethamine, cyclophosphamide, chlorambucil, melphalan, etc.),ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa),alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine,lomusitne, semustine, streptozocin, etc.), or triazenes (decarbazine,etc.). Examples of antimetabolites useful in the methods of the presentteachings include but are not limited to folic acid analog (e.g.,methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine,Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine,pentostatin). Examples of plant alkaloids and terpenoids or derivativesthereof include, but are not limited to, vinca alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine), podophyllotoxin, andtaxanes (e.g., paclitaxel, docetaxel). Examples of a topoisomeraseinhibitor include, but are not limited to, irinotecan, topotecan,amsacrine, etoposide, etoposide phosphate and teniposide. Examples ofantineoplastic agents include, but are not limited to, actinomycin,anthracyclines (e.g., doxorubicin, daunorubicin, valrubicin, idarubicin,epirubicin), bleomycin, plicamycin and mitomycin.

In one embodiment, the anti-cancer agents that can be used in thepresent teachings include Adriamycin, Dactinomycin, Bleomycin,Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;acronine; adoze le sin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;ifosfamide; ilmofosine; interleukin II (including recombinantinterleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b;interferon alfa-n1; interferon alfa-n3; interferon beta-I a; interferongamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate;letrozole; leuprolide acetate; liarozole hydrochloride; lometrexolsodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;my cophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Yet other anti-cancer agents/drugs that can be used in the presentteachings include, but are not limited to: 20-epi-1,25 dihydroxyvitaminD3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur;epirubicin; epristeride; estramustine analogue; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear poly amine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

In one embodiment, the anti-cancer agents that can be used in methodsdescribed herein are selected from the group consisting of paclitaxel,docetaxel, 5-fluorouracil, trastuzumab, lapatinib, bevacizumab,letrozole, goserelin, tamoxifen, cetuximab, panitumumab, gemcitabine,capecitabine, irinotecan, oxaliplatin, carboplatin, cisplatin,doxorubicin, epirubicin, cyclophosphamide, methotrexate, vinblastine,vincristine, melphalan and a combination thereof

In one embodiment, the anti-cancer agent and the compound represented byStructural Formula (I) are administered contemporaneously. Whenadministered contemporaneously, the anti-cancer agent and the compoundcan be administered in the same formulation or in differentformulations. Alternatively, the compound and the additional anti-canceragent are administered separately. Alternatively, the compound and theadditional anti-cancer agent can be administered sequentially, asseparate compositions, within an appropriate time frame (e.g., a cancertreatment session/interval (e.g., about 1.5 to about 5 hours to about 10hours to about 15 hours to about 20 hours; about 1 day to about 2 daysto about 5 days to about 10 days to about 14 days)) as determined by theskilled clinician (e.g., a time sufficient to allow an overlap of thepharmaceutical effects of the therapies). The compound and theadditional anti-cancer agent can be administered in a single dose ormultiple doses in an order and on a schedule suitable to achieve adesired therapeutic effect (e.g., inhibition of tumor growth).

In one embodiment, the subject in the methods described herein has notbeen previously treated with a TTK inhibitor (e.g., the compoundrepresented by Structural Formula (I).

The term “inhibiting the growth of tumor-initiating cells” refers todecreasing the rate of the proliferation and/or survival of thetumor-initiating cells.

As used herein, the term “reducing the likelihood of recurrence of acancer” means partially or totally inhibiting, delaying the return of acancer at or near a primary site and/or at a secondary site after aperiod of remission. It also means that the cancer is less likely toreturn with treatment described herein than in its absence.

As used herein, the term “remission” refers to a state of cancer,wherein the clinical symptoms or indicators associated with a cancerhave disappeared or cannot be detected, typically after the subject hasbeen successfully treated with an anti-cancer therapy.

As used herein, “treatment” is an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,reducing the likelihood of the spread of the disease, delay or slowingof disease progression, amelioration or palliation of the disease state,whether detectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.“Treatment” also includes reducing the likelihood of reoccurrence of thedisease.

As used herein, “treating a subject with a cancer” includes achieving,partially or substantially, one or more of the following: arresting thegrowth, reducing the extent of the cancer (e.g., reducing size of atumor), inhibiting the growth rate of the cancer, ameliorating orimproving a clinical symptom or indicator associated with the cancer(such as tissue or serum components) or increasing longevity of thesubject; and reducing the likelihood of recurrence of the cancer.

Generally, an effective amount of a compound taught herein variesdepending upon various factors, such as the given drug or compound, thepharmaceutical formulation, the route of administration, the type ofdisease or disorder, the identity of the subject or host being treated,and the like, but can nevertheless be routinely determined by oneskilled in the art. An effective amount of a compound of the presentteachings may be readily determined by one of ordinary skill by routinemethods known in the art.

The term an “effective amount” means an amount when administered to thesubject which results in beneficial or desired results, includingclinical results, e.g., inhibits, suppresses or reduces the cancer(e.g., as determined by clinical symptoms or the amount of cancer cells)in a subject as compared to a control.

In an embodiment, an effective amount of a compound taught herein rangesfrom about 0.1 to about 1000 mg/kg body weight, alternatively about 1 toabout 500 mg/kg body weight, and in another alternative, from about 20to about 300 mg/kg body weight. In another embodiment, an effectiveamount of a compound taught herein ranges from about 0.5 to about 5000mg/m², alternatively about from 5 to about 2500 mg/m², and in anotheralternative from about 50 to about 1000 mg/m². The skilled artisan willappreciate that certain factors may influence the dosage required toeffectively treat a subject suffering from cancer or reduce thelikelihood of recurrence of a cancer. These factors include, but are notlimited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject and otherdiseases present.

Moreover, for methods described herein (including treating a subjectwith a cancer or reducing the likelihood of recurrence of a cancer), a“treatment” or dosing regimen of a subject with an effective amount ofthe compound of the present teachings may consist of a singleadministration, or alternatively comprise a series of applications. Forexample, the compound of the present teachings may be administered atleast once a week. However, in another embodiment, the compound may beadministered to the subject from about one time per week to once dailyfor a given treatment. The length of the treatment period depends on avariety of factors, such as the severity of the disease, the age of thepatient, the concentration and the activity of the compounds of thepresent teachings, or a combination thereof. It will also be appreciatedthat the effective dosage of the compound used for the treatment mayincrease or decrease over the course of a particular treatment regime.Changes in dosage may result and become apparent by standard diagnosticassays known in the art. In some instances, chronic administration maybe required.

A “subject” is a mammal, preferably a human, but can also be an animalin need of veterinary treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, andthe like) and laboratory animals (e g., rats, mice, guinea pigs, and thelike).

The compounds taught herein can be administered to a patient in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. The compounds of thepresent teachings may be administered, for example, by oral, parenteral,buccal, sublingual, nasal, rectal, patch, pump or transdermaladministration and the pharmaceutical compositions formulatedaccordingly. Parenteral administration includes intravenous,intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal,intrapulmonary, intrathecal, rectal and topical modes of administration.Parenteral administration can be by continuous infusion over a selectedperiod of time.

The compounds taught herein can be suitably formulated intopharmaceutical compositions for administration to a subject. Thepharmaceutical compositions of the present teachings optionally includeone or more pharmaceutically acceptable carriers and/or diluentstherefor, such as lactose, starch, cellulose and dextrose. Otherexcipients, such as flavoring agents; sweeteners; and preservatives,such as methyl, ethyl, propyl and butyl parabens, can also be included.More complete listings of suitable excipients can be found in theHandbook of Pharmaceutical Excipients (5^(th) Ed., Pharmaceutical Press(2005)). A person skilled in the art would know how to prepareformulations suitable for various types of administration routes.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003—20th edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999. The carriers, diluents and/or excipients are“acceptable” in the sense of being compatible with the other ingredientsof the pharmaceutical composition and not deleterious to the recipientthereof.

Typically, for oral therapeutic administration, a compound of thepresent teachings may be incorporated with excipient and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

Typically for parenteral administration, solutions of a compound of thepresent teachings can generally be prepared in water suitably mixed witha surfactant such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, DMSO and mixturesthereof with or without alcohol, and in oils. Under ordinary conditionsof storage and use, these preparations contain a preservative to preventthe growth of microorganisms.

Typically, for injectable use, sterile aqueous solutions or dispersionof, and sterile powders of, a compound described herein for theextemporaneous preparation of sterile injectable solutions ordispersions are appropriate.

For nasal administration, the compounds of the present teachings can beformulated as aerosols, drops, gels and powders. Aerosol formulationstypically comprise a solution or fine suspension of the active substancein a physiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant which can be a compressed gassuch as compressed air or an organic propellant such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

For buccal or sublingual administration, the compounds of the presentteachings can be formulated with a carrier such as sugar, acacia,tragacanth, or gelatin and glycerine, as tablets, lozenges or pastilles.

For rectal administration, the compounds described herein can beformulated in the form of suppositories containing a conventionalsuppository base such as cocoa butter.

The compounds of invention may be prepared by methods known to thoseskilled in the art, as illustrated by the general schemes and proceduresbelow and by the preparative examples that follow. All startingmaterials are either commercially available or prepared by methods knownto those skilled in the art and the procedures described below.

In accordance with another aspect of the present invention, thecompounds of the invention can be prepared by processes analogues tothose established in the art. General methods to synthesize the claimedcompounds are elaborated below in Example A.

Exemplification EXAMPLE A Synthesis General Methods

Commercially available starting materials, reagents, and solvents wereused as received. In general, anhydrous reactions were performed underan inert atmosphere such as nitrogen or Argon. PoraPak® Rxn CX refers toa commercial cation-exchange resin available from Waters.

Microwave reactions were performed with a Biotage Initiator microwavereactor. Reaction progress was generally monitored by TLC using Mercksilica gel plates with visualization by UV at 254 nm, by analytical HPLCor by LCMS (Bruker Exquire 4000 or Waters Acquity UPLC system). Flashcolumn chromatographic purification of intermediates or final productswas performed using 230-400 mesh silica gel 60 from EMD chemicals orSilicycle, or purified using a Biotage Isolera with KP-SIL or HP-SILsilica cartridges, or KP-NH basic modified silica and correspondingsamplets. Reverse-phase HPLC purification was performed on a VarianPrepStar model SD-1 HPLC system with a Varian Monochrom 10μ C-18reverse-phase column using a of about 5-30% MeCN or MeOH/0.05% TFA-H₂Oto 70-90% MeCN or MeOH/0.05% TFA in H₂O over a 20-40-min period at aflow rate of 30-80 mL/min. Reverse phase purification was also performedusing a Biotage Isolera equipped with a KP-C18-H column using a between10-95% MeOH or CH₃CN/0.1% TFA in H₂O. Proton NMRs were recorded on aBruker 400 MHz spectrometer, and mass spectra were obtained using aBruker Esquire 4000 spectrometer or Waters Acquity UPLC system.

Compound names were generated using the software built intoCambridgeSoft-PerkinElmer's ChemBioDraw Ultra version 11.0 or 12.0.

Abbreviations:

-   aq aqueous-   Ar argon-   Boc tert-butoxycarbonyl-   br. broad-   calcd calculated-   d doublet-   DCM dichloromethane-   DIPEA diisopropylethylamine-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   dppf 1,1′-bis(diphenylphosphino) ferrocene-   h hour-   HPLC high performance liquid chromatography-   LC-MS liquid chromatography coupled to mass spectrometry-   min minute-   m multiplet-   MS ESI mass spectra, electrospray ionization-   NMR nuclear magnetic resonance-   O/N overnight-   PE petroleum ether-   PMB para-methoxybenzyl-   prep preparative-   rt room temperature-   s singlet-   t triplet-   TFA trifluoroacetic acid-   THF tetrahydrofuran

Intermediates:

4-bromo-N-cyclopropyl-2-methylbenzamide

To a suspension of 4-bromo-2-methylbenzoic acid (43.0 g, 200 mmol) andoxalyl dichloride (30.5 g, 240 mmol) in DCM (300 mL) was added DMF (0.1mL). The resulting reaction mixture was stirred at rt for 16 h. Thereaction turned into a clear yellow solution slowly over 16 h. Solventwas then removed in vacuo, and the crude product was used in the nextstep without further purification. The crude product was redissolved inDCM (300 mL) and cooled to 0° C. A mixture of TEA (42 mL, 300 mmol) andcyclopropylamine (12.6 g, 220 mmol) in DCM (100 mL) was added slowlyover 15 min, and the resulting mixture was stirred at rt for 2 h. Thereaction was diluted with DCM (200 mL) and water was added. Theresulting mixture was extracted with DCM and the combined organicextracts were dried over MgSO₄ and concentrated to give the desiredproduct as a pale pink solid (50.1 g, 99%). ¹H NMR (400 MHz, CDCl₃) δppm 7.37 (s, 1H), 7.31 (d, J=8.0 Hz, 1H), 7.17 (d, J=8.1 Hz, 1H), 5.93(br. s, 1H), 2.90-2.85 (m, 1H), 2.40 (s, 3H), 0.90-0.85 (m, 2H),0.62-0.58 (m, 2H); MS ESI [M+H]⁺ 253.9, calcd for [C₁₁H₁₂BrNO+H]⁺ 254.0.

N-(4-bromo-2-methylphenyl)cyclopropanecarboxamide

In a 100 mL RBF, 4-bromo-2-methylaniline (3.7 g, 20 mmol) and DIPEA(6.95 mL, 40 mmol) were combined with DMF (40 mL). The reaction wascooled to 0° C. in an ice bath and cyclopropanecarbonyl chloride (2.1 g,20 mmol) was added. The mixture was stirred at 0° C. for 1 h. Water andEtOAc were added to separate the phases and the aqueous phase wasextracted with EtOAc. The organic layers were combined, dried overNa₂SO₄, filtered, and concentrated under reduced pressure to give thetitle compound as a white solid (4.76 g, 94%). ¹H NMR (400 MHz, CDCl₃) δppm 7.85-7.72 (m, 1H), 7.38-7.28 (m, 2H), 7.15-7.02 (m, 1H), 2.27 (s,3H), 1.57-1.48 (m, 1H), 1.10 (quint, J=3.9 Hz, 2H), 0.92-0.79 (m, 2H);MS ESI [M+H]⁺ 253.9, calcd for [C₁₁H₁₂BrNO+H]⁺ 254.0.

N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide

To a mixture of 4-bromo-N-cyclopropyl-2-methylbenzamide (3.73 g, 14mmol), Bis(pinacolato)diboron (5.59 g, 22 mmol), anh KOAc (4.29 g, 43mmol) in DMF (37 mL) was purged with Ar for 10 min at rt. ThenPdCl₂(dppf)·DCM (0.59 g, 5 mol %) was added and the reaction was heatedat 100° C. in oil bath for 4 h. After reaction completion the reactionmass was diluted with EtOAc (200 mL) and H₂O (100 mL). The combinedlayer filtered through celite pad and washed it with little EtOAc. Theaq. layer further extracted with EtOAc (50 mL) and the combined organiclayer washed with brine, dried over Na₂SO₄, filtered, and concentratedto give crude oily residue. The crude product was purified by flashchromatography (gradient: EtOAc/hex 0-100%) to give the title compoundas a creamy solid (4.15 g, 94%). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.66 (s,1H), 7.63-7.61 (d, J=7.6 Hz, 1H), 7.32-7.30 (d, J=7.6 Hz, 1H), 5.85 (s,1 H), 2.93-2.87 (m, 1H), 2.46 (s, 3H), 1.35 (s, 12H), 0.90-0.86 (m, 2H),0.63-0.59 (m, 2H); MS ESI [M+H]⁺ 302.2, calcd for [C₁₇H₂₄BNO₃+H]⁺ 302.2.

3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine

To a stirred solution of sodium ethoxide in EtOH, which was preparedfrom sodium (281.3 g, 12.0 mol) and EtOH (10 L) by the conventionalmethod, were added diethyl malonate (963.7 g, 6.02 mol) at ambienttemperature and then compound 1H-pyrazol-3-amine (500 g, 6.02 mol). Thereaction mixture was refluxed for 12 hours. After cooled to roomtemperature, the precipitates were collected by filtration and dissolvedin water. The aqueous solution was acidified with 2 M HCl (pH=2). Theresulting precipitates were collected by filtration and dried underreduced pressure to afford pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione(649 g, 71%) as a yellow solid, which was used for the next reactionwithout further purification.

A stirred suspension of pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (265g, 1.75 mol) and N,N-dimethylaniline (335.6 mL) in POCl₃ (2.00 kg, 13.2mol) was refluxed for 4 hours. After cooled to room temperature, thereaction mixture was poured into ice-water, and stirred for 30 min,neutralized with saturated aqueous sodium carbonate and extracted withEtOAc. The combined organic layers were washed with water, brine anddried over MgSO₄, filtered and evaporated. The residue was purified bycolumn chromatography on silica gel (gradient: EtOAc/PE 1:10) to give5,7-dichloropyrazolo[1,5-a]pyrimidine (287 g, 87%) as a yellow solid.

To a solution 5,7-dichloropyrazolo[1,5-a]pyrimidine (246.6 g, 1.31 mol)in CH₃CN (1.8 L) was added NBS (245 g, 1.38 mol). The resulting mixturewas stirred at room temperature for 2 hours. After removal of thesolution, the reaction mixture was purified by column chromatography onsilica gel (gradient:EtOAc/PE 1:5) to give the title compound (313.5 g,89%) as light yellow solid. ¹H NMR (300 MHz, CDCl₃): δ ppm 7.04 (s, 1H),8.21 (s, 1H); MS ESI [M+H]⁺ 265.9, calcd for [C₆H₂BrCl₂N₃+H]⁺ 265.9.

3-bromo-5-chloro-N-(cyclopropylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine

To a solution of 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine (5.0 g,19 mmol) in DCM (50 mL) was added cyclopropylmethanamine (1.48 g, 21mmol), and DIPEA (6.6 mL, 38 mmol). The reaction was stirred at rt for 2h. Water and DCM were added to separate the phases and the aqueous phasewas extracted with DCM. The combined organic extracts were dried overNaSO₄, filtered and concentrated. The crude product was purified byflash chromatography (gradient: EtOAc/hex 0-50%) to give the titlecompound as a yellow solid (5.25 g, 92%). ¹H NMR (400 MHz, CDCl₃) δ ppm7.97 (s, 1H), 6.50 (br. s, 1H), 5.97 (s, 1H), 3.25 (dd, J=7.3, 5.5 Hz,2H), 1.26-1.13 (m, 1H), 0.74-0.65 (m, 2H), 0.37 (q, J=5.0 Hz, 2H); MSESI [M+H]⁺ 301.0, calcd for [C₁₀H₁₀BrClN₄+H]⁺ 301.0.

(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)-1-methylcyclobutanol

To a solution of 3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine (14.6 g,55.3 mmol) in DCM (200 mL) was addedcis-hydroxy-3-methylcyclobutane-1-methylamine (7.0 g, 60.9 mmol), andDIPEA (19.2 mL, 110.6 mmol). The reaction was stirred at rt for 16 h.Water and DCM were added to separate the phases and the aqueous phasewas extracted with DCM. The combined organic extracts were dried overNa₂SO₄, filtered and concentrated to give the title compound as a yellowsolid (18.1 g, 95%). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.96 (s, 1H),6.60-6.49 (m, 1H), 5.99 (s, 1H), 3.47 (t, J=6.0 Hz, 2H), 2.38-2.27 (m,3H), 1.96-1.85 (m, 2H), 1.43 (s, 3H); MS ESI [M+H]⁺ 345.1, calcd for[C₁₂H₁₄BrClN₄O+H]⁺ 345.0.

tert-butyl(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate

To a solution of3-bromo-5-chloro-N-(cyclopropylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine(5.25 g, 17.5 mmol) in DCM (150 mL) was added Boc₂O (5.70 g, 26.2 mmol),DMAP (0.21 g, 1.75 mmol), and TEA (7.3 mL, 52.5 mmol). The reaction wasstirred at rt for 4 h. Water and DCM were added to separate the phasesand the aqueous phase was extracted with DCM. The combined organicextracts were dried over MgSO₄, filtered and concentrated. The crudeproduct was purified by flash chromatography (gradient: EtOAc/hex 0-40%)to give the title compound as a yellow solid (6.24 g, 89%). ¹H NMR (400MHz, CDCl₃) δ ppm 8.12 (s, 1H), 6.85 (s, 1H), 3.72 (d, J=7.3 Hz, 2H),1.39 (s, 9H), 1.05-0.94 (m, 1H), 0.46-0.37 (m, 2H), 0.13-0.03 (m, 2H);MS ESI [M-C₄H₈]⁺ 345.0, calcd for [C₁₅H₁₈BrClN₄O₂—C₄H₈]⁺ 345.0.

tert-butyl(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate

To a solution of(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)-1-methylcyclobutanol(18.1 g, 52.6 mmol), Boc₂O (34.3 g, 158 mmol), and), and TEA (22 mL,157.8 mmol) in DCM (200 mL) was added DMAP (1.28 g, 10.5 mmol). Thereaction was stirred at 40° C. for 16 h. The solvent was removed invacuo and water and DCM were added to separate the phases and theaqueous phase was extracted with DCM. The combined organic extracts weredried over NaSO₄, filtered and concentrated. The crude product waspurified by flash chromatography (3 Biotage 100 g SiO₂ columns inparallel, gradient: EtOAc/hex 0-30%) to give the title compound as abeige solid (12.7 g, 44%). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.11 (s, 1H),6.74 (s, 1H), 3.88 (d, J=6.5 Hz, 2H), 2.17 (s, 3H), 1.99-1.88 (m, 2H),1.47 (s, 3H), 1.45 (s, 9H), 1.35 (s, 9H); MS ESI [M+H]⁺ 545.1, calcd for[C₂₂H₃₀BrClN₄O₅+H]⁺ 545.1.

(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenzyl)amino)methyl)-1-methylcyclobutanol

To a solution of(1s,3s)-3-(((3-bromo-5-chloropyrazol[1,5-a]pyrimidin-7-yl)amino)methyl)-1-methylcyclobutanol(12.0 g, 34.7 mmol) and 4-methoxybenzyl chloride (5.2 mL, 38.2 mmol) inDMF (50 mL) was added K₂CO₃ (9.6 g, 69.4 mmol). The resulting mixturewas stirred at 60° C. for 3 h. Water and EtOAc were added to separatethe phases and the aqueous phase was extracted with EtOAc. The combinedorganic extracts were washed with water, dried over MgSO₄, filtered andconcentrated. The crude product was purified by flash chromatography(gradient: EtOAc/hex 10-100%) using three columns in parallel. Impurefractions were collected and purified by flash chromatography using theabove conditions. Pure fractions were combined and concentrated to givethe desired product as a pale yellow solid (13.1 g, 81%). ¹H NMR (400MHz, CDCl₃) δ ppm 8.01 (s, 1H), 7.17 (d, J=8.7 Hz, 2H), 6.67 (d, J=8.7Hz, 2H), 6.02 (s, 1H), 4.98 (s, 2H), 3.83 (d, J=6.4 Hz, 2H), 3.81 (s,3H), 2.25-2.16 (m, 3H), 1.76-1.72 (m, 2H), 1.36 (s, 3H); MS ESI [M+H]⁺467.1, calcd for [C₂₀H₂₂BrClN₄O₂+H]+ 467.1.

tert-butyl(3-bromo-5-(((1S,2R)-2-hydroxycyclohexyl)amino)pyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate

To a solution of using tert-butyl(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate(9.0 g, 22.5 mmol) in NMP (90 mL) was added(1R,2S)-2-aminocyclohexanol—HCl (4.08 g, 27.0 mmol), and DIPEA (3.47 g,25.1 mmol). The reaction was divided and sealed into six microwavevials. Each vial was microwaved for 3 h at 130° C. Water and EtOAc wereadded to separate the phases and the aqueous phase was extracted withEtOAc. The combined organic extracts were dried over Na₂SO₄, filteredand concentrated. The crude product was purified by flash chromatography(gradient: EtOAc/hex 0-50%) using three columns in parallel to give thetitle compound as a beige solid (8.51 g, 79%). ¹H NMR (400 MHz, CDCl₃) δppm 7.80 (s, 1H), 6.08 (s, 1H), 5.33-5.19 (m, 1H), 4.26-4.14 (m, 1H),4.13-4.06 (m, 1H), 3.61 (d, J=7.3 Hz, 2H), 2.51 (br. s, 1H), 1.89-1.62(m, 8H), 1.55-1.45 (m, 2H), 1.40 (s, 9H), 1.06-0.95 (m, 1H), 0.47-0.38(m, 2H), 0.17-0.07 (m, 2H); MS ESI [M+H]⁺ 408.3, calcd for[C₂₁H₃₀BrN₅O₃+H]⁺ 480.2.

tert-butyl(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate

A mixture of tert-butyl(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate(400 mg, 1.0 mmol), 3-hydroxypyridine (475 mg, 5.0 mmol), DBU (0.75 mL,5.0 mmol) in DME (5 mL) was combined and sealed into a microwave vial.The mixture was microwaved for 1 h at 85° C. Water and EtOAc were addedto separate the phases and the aqueous phase was extracted with EtOAc.The combined organic extracts were dried over Na₂SO₄, filtered andconcentrated. The crude product was purified by flash chromatography(gradient: EtOAc/hex 0-50%) to give the title compound as a brown oil(367 mg, 80%). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.64 (d, J=2.5 Hz, 1H) 8.54(dd, J=4.5, 1.5 Hz, 1H) 7.97 (s, 1H), 7.78-7.72 (m, 1H), 7.44-7.38 (m,1H) 6.61 (s, 1H) 3.71 (d, J=7.3 Hz, 2H) 1.39 (s, 9 H), 1.06-1.01 (m, 1H)0.45 (s, 2H), 0.13-0.09 (m, 2H); MS ESI [M+H]⁺ 460.0, calcd for[C₂₀H₂₂BrN₅O₃+H]⁺ 460.1.

tert-butyl(3-bromo-5-(cyclopentylamino)pyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate

To a solution of tert-butyl(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate(0.45 g, 0.83 mmol) in NMP (4 mL) was added cyclopentylamine (0.085 g,1.0 mmol), and DIPEA (0.21 g, 1.66 mmol). The reaction was microwavedfor 3 h at 130° C. Water and EtOAc were added to separate the phases andthe aqueous phase was extracted with EtOAc. The combined organicextracts were dried over Na₂SO₄, filtered and concentrated. The crudeproduct was purified by flash chromatography (gradient: EtOAc/hex 0-60%)to give the title compound as a white solid (0.40 g, 67%). ¹H NMR (400MHz, CDCl₃) δ ppm 7.79 (s, 1H), 5.94 (s, 1H), 5.08 (br. s, 1H), 4.23(br. s, 1H), 3.85-3.72 (m, 2H), 2.28-2.06 (m, 5H), 2.02-1.89 (m, 2H),1.81-1.61 (m, 5H), 1.48 (s, 4H), 1.44 (s, 9H), 1.36 (s, 9H); MS ESI[M+H]⁺ 594.3, calcd for [C₂₇H₄₀BrN₅O₅+H]⁺ 594.2.

tert-butyl(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate

A mixture of tert-butyl(3-bromo-5-chloropyrazol[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate(12.7 g, 23.3 mmol), 3-hydroxypyridine (4.43 g, 46.6 mmol), and K₂CO₃(9.65 g, 69.9 mmol) was combined in DMF (100 mL) and stirred at rtovernight. The reaction mixture was diluted with EtOAc and washed withwater, dried over Na₂SO₄, filtered and concentrated in vacuo. The crudeproduct was triturated with Et₂O and filtered to give the title compoundas a beige solid (8.43 g, 14.0 mmol). The mother liquor was concentratedin vacuo and purified by flash chromatography (gradient: EtOAc/hex0-40%) to give the title compound as a light orange solid (4.07 g, 6.74mmol). The solids obtained through trituration and flash chromatographywere combined to give the title compound as a light orange solid (12.50g, 89%) ¹H NMR (400 MHz, CDCl₃) δ ppm 8.65 (d, J=2.8 Hz, 1H), 8.55 (dd,J=4.6, 1.4 Hz, 1H), 7.98 (s, 1H), 7.78-7.73 (m, 1H), 7.46-7.40 (m, 1H),6.50 (s, 1H), 3.89 (d, J=6.8 Hz, 2H), 2.30-2.15 (m, 3H), 2.02-1.93 (m,2H), 1.50 (s, 3H), 1.45 (s, 9H), 1.38 (s, 9H); MS ESI [M+H]⁺ 604.3,calcd for [C₂₇H₃₄BrN₅O₆+H]⁺ 604.2.

(1s,3s)-3-(((3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenzyl)amino)methyl)-1-methylcyclobutanol

To a solution of(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenzyl)amino)methyl)-1-methylcyclobutanol(13.1 g, 28.2 mmol) and 3-hydroxypyridine (4.0 g, 42.3 mmol) in DMF (70mL) was added K₂CO₃ (7.8 g, 56.3 mmol). The resulting mixture wasstirred at 80° C. for 22 h. A mixture of 60% NaH (0.6 g, 15 mmol) and3-hydroxypyridine (1.4 g, 14.7 mmol) in DMF was stirred at rt for 30 minbefore adding to the reaction mixture. The resulting solution wasstirred at 100° C. for 20 h. Water and EtOAc were added to separate thephases and the aqueous phase was extracted with EtOAc. The combinedorganic extracts were washed with water, dried over MgSO₄, filtered andconcentrated. The crude product was purified by flash chromatography(gradient: EtOAc/hexanes 10-100%) using three columns in parallel.Impure fractions were collected and purified by flash chromatography(gradient: EtOAc/hexanes 50-100%). Pure fractions were combined andconcentrated to give the desired product as a pale yellow solid (9.5 g,64%). H NMR (400 MHz, CDCl₃) δ ppm 8.56 (d, J=4 Hz, 1H), 8.45 (d, J=8Hz, 1H), 8.00 (s, 1H), 7.69-7.65 (m, 1H), 7.36 (dd, J=4.8, 0.4 Hz, 1H),7.19 (d, J=8.7 Hz, 2H), 6.85 (d, J=8.7 Hz, 2H), 5.71 (s, 1H), 4.91 (s,2H), 3.87 (d, J=6.7 Hz, 2H), 3.87 (s, 3H), 2.30-2.15 (m, 3H), 1.77 (t,J=8.1 Hz, 2H), 1.37 (s, 3H); MS ESI [M+H]⁺ 524.2, calcd for[C₂₅H₂₆BrN₅O₃+H]+ 524.1.

N-cyclopropyl-4-(7-((((1s,3s)-3-hydroxy-3-methylcyclobutyl)methyl)(4-methoxybenzyl)amino)-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamide

A mixture of(1s,3s)-3-(((3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenzyl)amino)methyl)-1-methylcyclobutanol(9.5 g, 18.1 mmol),N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide(7.6 g, 25.4 mmol), 2M K₃PO₄ (23 mL, 45.3 mmol) in THF (100 mL) waspurged with Ar for 10 min before the addition of PdCl₂dppfDCM (1.5 g,1.8 mmol). The resulting mixture was heated under reflux in an oil bathat 84° C. for 16 h. The reaction mixture was diluted with DCM and sat.NaHCO₃. The aqueous phase was extracted with DCM and the combinedorganic extracts were dried over MgSO₄, filtered and concentrated. Thecrude product was purified by flash chromatography (gradient: EtOAc/hex50-100%) using three columns in parallel. Impure fractions werecollected and purified by flash chromatography using the aboveconditions. Pure fractions were combined and concentrated to give thedesired product as an orange solid (9.6 g, 85%). 1H NMR (400 MHz, CDCl₃)δ ppm 8.90-8.84 (m, 1H), 8.55 (d, J=4.5 Hz, 1H), 8.29 (s, 1H). 7.88-7.83(m, 1H), 7.63 (s, 1H), 7.58-7.53 (m, 2H), 7.35-7.31 (m, 1H), 7.23 (d,J=8.6 Hz, 2H), 6.89 (d, J=8.6 Hz, 2H), 6.19 (br. s, 1H), 5.82 (s, 1H),4.99 (s, 2H), 3.90 (d, J=10.0 Hz, 2H), 3.80 (s, 3H), 2.93-2.90 (m, 1H),2.40 (s, 3H), 2.36-2.28 (m, 1H), 2.22-2.20 (m, 2H), 1.89-1.88 (m, 1H),1.80 (t, J=8.7 Hz, 2H), 1.38 (s, 3H), 0.88-0.85 (m, 2H), 0.68-0.64 (m,2H); MS ESI 619.3 [M+H]⁺, calcd for [C₃₆H₃₈N₆O₄+H]+ 619.3.

Preparation of Exemplary Compounds of the Invention A1:N-cyclopropyl-4-(7-((cyclopropylmethyl)amino)-5-(((1S,2R)-2-hydroxycyclohexyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamidehydrochloride

A mixture of tert-butyl(3-bromo-5-(((1S,2R)-2-hydroxycyclohexyl)amino)pyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate(8.48 g, 17.7 mmol),N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide(6.92 g, 23.0 mmol), PdCl₂dppfDCM (1.44 g, 1.76 mmol), and 2M K₃PO₄(26.6 mL, 106.2 mmol) in THF (60 mL) was divided and sealed into fourmicrowave vials. Each vial was charged with Ar and heated in themicrowave at 125° C. for 3 h. Water and EtOAc were added to separate thephases and the aqueous phase was extracted with EtOAc. The combinedorganic extracts were dried over Na₂SO₄, filtered and concentrated. Thecrude product was purified by flash chromatography (gradient: EtOAc/hex20-100%) using two columns in parallel to give a yellow solid.

The above compound was dissolved in DCM (20 mL) and treated with TFA (20mL) at rt for 1 h. After reaction completion, solvent was removed invacuo and the crude was redissolved in DCM (20 mL), neutralized withsaturated aqueous sodium bicarbonate and extracted with EtOAc. Thecombined organic extracts were dried over NaSO₄, filtered andconcentrated. The crude product was purified by flash chromatography(gradient: 50-100% EtOAc/hex then MeOH/DCM 0-10%) and triturated withEt₂O to give the title compound as a free base (white solid). The freebase was dissolved in a mixture of DCM (25 mL) and MeOH (50 mL), HCl (1MEt₂O, 2 equiv) was then added slowly. Solvent was removed in vacuo togive the title compound as a pale yellow solid in HCl salt (2.09 g, 25%over 2 steps). ¹H NMR (400 MHz, CD₃OD) δ ppm 8.21 (s, 1H), 7.51-7.37 (m,3H), 5.67 (br. s, 1H), 4.04-3.82 (m, 2H), 3.46-3.37 (m, 2H), 2.93-2.82(m, 1H), 2.47 (s, 3H), 1.89-1.64 (m, 6H), 1.61-1.42 (m, 2H), 1.31-1.21(m, 1H), 0.88-0.78 (m, 2H), 0.70-0.58 (m, 4H), 0.46-0.36 (m, 2H); MS ESI[M+H]⁺ 475.3, calcd for [C₂₇H₃₄N₆O₂+H]⁺ 475.3. HPLC purity: 98% at 254nm.

A2:N-cyclopropyl-4-(7-((cyclopropylmethyl)amino)-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamidehydrochloride

A mixture of tert-butyl(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbamate(367 mg, 0.80 mmol)),N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide(289 mg, 0.96 mmol), PdCl₂dppfDCM (65 mg, 0.080 mmol), and 2M K₃PO₄ (1.4mL, 2.8 mmol) in THF (4 mL) was sealed into a microwave vial. The vialwas charged with Ar and heated in the microwave at 130° C. for 4 h.Water and EtOAc were added to separate the phases and the aqueous phasewas extracted with EtOAc. The combined organic extracts were dried overNa₂SO₄, filtered and concentrated. The crude product was purified byflash chromatography (gradient: EtOAc/hex 20-100%) to give a yellow oil.

The above compound was dissolved in DCM (6 mL) and treated with TFA (2mL) at rt for 1 h. After reaction completion, solvent was removed invacuo and the crude was redissolved in MeOH (4 mL), filtered, andpurified by prep HPLC. The fractions were passed through a PoraPakcolumn and triturated with Et₂O to give the title compound as a freebase (white solid, 119 mg). A portion of the free base (67 mg, 0.15mmol) was then dissolved in MeOH (10 mL), and HCl (1M Et₂O, 2 equiv) wasthen added slowly. The mixture was concentrated and triturated with Et₂Oto give the title compound as a yellow solid in HCl salt (55 mg, 25%yield over 2 steps). ¹H NMR (400 MHz, CD₃OD) δ ppm 9.10 (s, 1H), 8.82(d, J=6.0 Hz, 1H), 8.73-8.66 (m, 1H), 8.41 (s, 1H), 8.24-8.15 (m, 1H),7.69 (s, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 6.07 (s,1H), 3.39 (d, J=7.0 Hz, 2H), 2.89-2.78 (m, 1H), 2.29 (s, 3H), 1.37-1.23(m, 1H), 0.86-0.75 (m, 2H), 0.70-0.56 (m, 4H), 0.45-0.38 (m, 2H); MS ESI[M+H]⁺ 455.2, calcd for [C₂₆H₂₆N₆O₂+H]⁺ 455.2. HPLC purity: 95.9% at 254nm.

A3:4-(5-(cyclopentylamino)-7-((((1s,3s)-3-hydroxy-3-methylcyclobutyl)methyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-N-cyclopropyl-2-methylbenzamidehydrochloride

A mixture of tert-butyl(3-bromo-5-(cyclopentylamino)pyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate(0.40 g, 0.67 mmol),N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide(0.26 g, 0.88 mmol), PdCl₂dppfDCM (0.055 g, 0.067 mmol), and 2M K₃PO₄ (1mL, 2.01 mmol) in THF (4 mL) was charged with Ar and heated in themicrowave at 130° C. for 3 h. Water and EtOAc were added to separate thephases and the aqueous phase was extracted with EtOAc. The combinedorganic extracts were dried over Na₂SO₄, filtered and concentrated. Thecrude product was purified by flash chromatography (gradient: EtOAc/hex20-100%) to give a yellow oil.

The above compound was dissolved in DCM (10 mL) and treated with TFA (3mL) at rt for 3 h. After reaction completion, solvent was removed invacuo and the crude product was dissolved in MeOH (5 mL). The mixturewas filtered and purified by prep-HPLC. The compound was passed througha PoraPak cartridge and triturated with Et₂O to give the title compoundas a free base (white solid). The free base was dissolved in MeOH (5mL), and HCl (1M Et₂O, 2 equiv) was then added slowly. Solvent wasremoved in vacuo to give the title compound as a light orange solid inHCl salt (75 mg, 21% over 2 steps). ¹H NMR (400 MHz, CD₃OD) δ ppm 8.18(s, 1H), 7.42 (s, 3H), 5.52 (br. s, 1H), 4.23-4.11 (m, 1H), 3.68-3.54(m, 2H), 2.93-2.81 (m, 1H), 2.46 (s, 3H), 2.38-2.27 (m, 1H), 2.26-2.07(m, 4H), 1.99-1.89 (m, 2H), 1.88-1.59 (m, 6H), 1.35 (s, 3H), 0.86-0.79(m, 2H), 0.66-0.58 (m, 2H); MS ESI [M+H]⁺ 489.4, calcd for[C₂₈H₃₆N₆O₂+H]⁺ 489.3. HPLC purity: 99% at 254 nm.

A4:N-cyclopropyl-4-(7-((((1s,3s)-3-hydroxy-3-methylcyclobutyl)methyl)amino)-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamidehydrochloride and its free base

A). Through Boc deprotection: A mixture of tert-butyl(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate(0.23 g, 0.38 mmol),N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide(0.15 g, 0.49 mmol), PdCl₂dppfDCM (0.15 g, 0.49 mmol), and 2M K₃PO₄(0.57 mL, 1.14 mmol) in THF (4 mL) was charged with Ar and heated in themicrowave at 130° C. for 3 h. Water and EtOAc were added to separate thephases and the aqueous phase was extracted with EtOAc. The combinedorganic extracts were dried over NaSO₄, filtered and concentrated. Thecrude product was purified by flash chromatography (gradient: EtOAc/hex20-60%) to give a yellow oil.

The above intermediate was dissolved in DCM (10 mL) and treated with TFA(3 mL) at rt for 3 h. After reaction completion, solvent was removed invacuo and the crude product was dissolved in MeOH (5 mL). The mixturewas filtered and purified by prep-HPLC. The compound was passed througha PoraPak cartridge and triturated with Et₂O to give the title compoundas a free base (white solid). The free base was dissolved in MeOH (5mL), and HCl (1 M Et₂O, 2 equiv) was then added slowly. Solvent wasremoved in vacuo to give the title compound as a beige solid in HCl salt(96 mg, 47% over 2 steps). ¹H NMR (400 MHz, CD₃OD) δ ppm 9.14 (br. s,1H), 8.89-8.82 (m, 1H), 8.79-8.71 (m, 1H), 8.40 (s, 1H), 8.31-8.21 (m,1H), 7.68 (s, 1H), 7.59 (d, J=9.5 Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 6.06(s, 1H), 3.56 (d, J=6.5 Hz, 2H), 2.88-2.79 (m, 1H), 2.40-2.31 (m, 1H),2.29 (s, 3H), 2.26-2.18 (m, 2H), 1.99-1.89 (m, 2H), 1.37 (s, 3H),0.85-0.76 (m, 2H), 0.63-0.53 (m, 2H); MS ESI [M+H]⁺ 499.3, calcd for[C₂₈H₃₀N₆O₃+H]⁺ 499.2. HPLC purity: 99.5% at 254 nm.

B). Through PMB deprotection: A mixture ofN-cyclopropyl-4-(7-((((1s,3s)-3-hydroxy-3-methylcyclobutyl)methyl)(4-methoxybenzyl)amino)-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamide(9.6 g, 15.5 mmol), TFA (50 mL) in DCE (70 mL) was heated in an oil bathat 50° C. for 4 h. After reaction completion, solvent was removed invacuo and the crude product was dissolved in a mixture of MeOH/DCM (100mL/25 mL). 2M Na₂CO₃ (150 mL) was then added and the resulting mixturewas stirred at rt for 30 min. The reaction mixture was diluted with DCMand the phases were separated. The aqueous phase was extracted with DCMand the combined organic extracts were washed with water, dried overMgSO₄, filtered and concentrated. The crude product was triturated andsonicated in a mixture of DCM/Et₂O (10 mL/70 mL) to give the titlecompound as a off white solid in free base (5.9 g, 77%). ¹H NMR (400MHz, CD₃OD) δ ppm 8.58-8.53 (m, 1H), 8.50-8.46 (m, 1H), 8.36 (s, 1H),7.86-7.80 (m, 1H), 7.76-7.72 (m, 1H), 7.61-7.55 (m, 2H), 7.18 (d, J=8.0Hz, 1H), 5.92 (s, 1H), 3.52 (d, J=6.8 Hz, 2H), 2.86-2.77 (m, 1H),2.38-2.28 (m, 1H), 2.25 (s, 3H), 2.24-2.18 (m, 2H), 1.99-1.88 (m, 2H),1.37 (s, 3H), 0.84-0.75 (m, 2H), 0.64-0.54 (m, 2H); MS ESI [M+H]⁺ 499.2,calcd for [C₂₈H₃₀N₆O₃+H]⁺ 499.2. HPLC purity: 96.1% at 235 nm.

The following final compounds were synthesized similarly to thesyntheses of A1-4.

Example number MS calcd; MS ESI [M + H]⁺ Salt Form; Structure HPLCpurity ¹H NMR

A5  [C₂₈H₃₀N₆O₃ + H]⁺ 499.2; 499.3; free base; 98.9% at 254 nM (400 MHz,CD₃OD) δ ppm 8.55 (dd, J = 2.8, 0.4 Hz, 1H), 8.47 (dd, J = 4.8, 1.6 Hz,1H), 8.33 (s, 1 H), 7.83-7.80 (m, 1H), 7.72 (s, 1H), 7.58-7.55 (m, 2H),7.18 (d, J = 8.0 Hz, 1H), 5.89 (s, 1H), 3.49 (d, J = 7.6 Hz, 2H),2.87-2.78 (m, 2H), 2.28-2.23 (m, 5H), 1.97-1.92 (m, 2H), 1.36 (s, 3H),0.82-0.77 (m, 2H), 0.62-0.58 (m, 2H).

A6  [C₂₇H₂₈N₆O₃ + H]⁺ 485.2; 485.3; HCl salt; 98.8% at 254 nm (400 MHz,CD₃OD) δ ppm 9.13 (d, J = 2.3 Hz, 1H), 8.86 (d, J = 5.8 Hz, 1H),8.77-8.69 (m, 1H), 8.40 (s, 1H), 8.26 (dd, J = 8.8, 5.8 Hz, 1H), 7.67(s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 6.07 (s,1H), 4.53-4.39 (m, 1H), 3.57 (d, J = 8.0 Hz, 2H), 2.89-2.79 (m, 1H),2.76-2.61 (m, 1H), 2.30 (s, 3H), 2.29-2.21 (m, 2H), 2.21-2.08 (m, 2H),0.85-0.78 (m, 2H), 0.65-0.58 (m, 2H).

A8  [C₂₇H₃₄N₆O₂ + H]+ 475.3; 475.4; HCl salt; 99.3% at 254 nm (400 MHz,CD₃OD) δ ppm 8.22 (s, 1H), 7.51-7.36 (m, 3H), 5.68 (s, 1H), 3.77 (s,2H), 3.37-3.33 (m, 2H), 2.93-2.84 (m, 1H), 2.47 (s, 3H), 2.07-1.95 (m,4H), 1.93-1.72 (m, 4H), 1.34-1.19 (m, 1H), 0.88-0.78 (m, 2H), 0.69-0.59(m, 4H), 0.45-0.37 (m, 2H).

A11 [C₂₇H₂₈N₆O₃ + H]⁺ 485.2  485.2; HCl salt; 98.6% at 254 nm (400 MHz,CD₃OD) δ ppm 9.14 (s, 1H), 8.85 (d, J = 5.5 Hz, 1H), 8.79-8.72 (m, 1H),8.40 (s, 1H), 8.30-8.21 (m, 1H), 7.68 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H),7.23 (d, J = 8.0 Hz, 1H), 6.06 (s, 1H), 4.17-4.06 (m, 1H), 3.54 (d, J =6.5 Hz, 2H), 2.88-2.78 (m, 1H), 2.53-2.43 (m, 2H), 2.29 (s, 4H),1.81-1.68 (m, 2H), 0.84-0.77 (m, 2H), 0.62-0.56 (m, 2H).

A12 [C₂₇H₃₄N₆O₂ + H]⁺ 475.3; 475.4; HCl salt; 97.5% at 254 nm (400 MHz,CD₃OD) δ ppm 8.18 (s, 1H), 7.55-7.29 (m, 3H), 5.54 (br, s, 1H),4.50-4.37 (m, 1H), 4.26-4.07 (m, 1H), 3.62 (d, J = 6.8 Hz, 2H),2.92-2.83 (m, 1H), 2.75-2.61 (m, 1H), 2.46 (s, 3H), 2.31-2.02 (m, 6H),1.92-1.56 (m, 6H), 0.91-0.75 (m, 2H), 0.71-0.52 (m, 2H).

A13 [C₂₈H₃₆N₆O₂ + H]⁺ 489.3; 489.4  TFA salt; 98.2% at 254 nm (400 MHz,CD₃OD) δ ppm 8.18 (s, 1H), 7.48-7.36 (m, 3H), 5.52 (s, 1H), 4.23-4.10(m, 1H), 3.65-3.57 (m, 2H), 2.93-2.75 (m, 2H), 2.46 (s, 3H), 2.31-2.21(m, 2H), 2.20-2.11 (m, 3H), 2.06-1.89 (m, 2H), 1.89-1.50 (m, 6H), 1.37(s, .3H), 0.87-0.79 (m, 2H), 0.68-0.56 (m, 2H).

EXAMPLE B TTK Inhibition Assay

Active TTK was purchased from Invitrogen as an amino terminal GST fusionof full length human TTK. Amino terminal 6 histidine, sumo tagged humanTTK (residues 1-275) was expressed in E. coli, and purified to >95%homogeneity by Ni²⁺ agarose, gel filtration, and ion exchangechromatography.

TTK activity was measured using an indirect ELISA detection system.GST-TTK (0.68 nM) was incubated in the presence of either 16 μM ATP(Sigma cat#A7699) or 100 μM ATP, 50 mM Hepes pH 7.2, 1 mM EGTA, 10 mMMgCl₂, and 0.1% Pluronic in a 96 well microtitre plate pre-coated withamino terminal 6 histidine, sumo tagged TTK (amino acid residues 1-275).

The reaction was allowed to proceed for 30 minutes, followed by 5 washesof the plate with Wash Buffer (phosphate buffered saline supplementedwith 0.2% Tween 20), and incubation for 30 minutes with a 1:3000dilution of primary antibody (Cell Signaling cat#9381). The plate waswashed 5 times with Wash Buffer, incubated for 30 minutes in thepresence of secondary antibody coupled to horse radish peroxidase(BioRad cat#1721019, 1:3000 concentration), washed an additional 5 timeswith Wash Buffer, and incubated in the presence of TMB substrate (Sigmacat#T0440). The colourimetric reaction was allowed to continue for 5minutes, followed by addition of stop solution (0.5 N sulphuric acid),and quantified by detection at 450 nm with either a monochromatic orfilter based plate reader (Molecular Devices M5 or Beckman DTX880,respectively).

Compound inhibition was determined at either a fixed concentration (10μM) or at a variable inhibitor concentration (typically 0.5 μM to 0.001μM in a 10 point dose o response titration). Compounds werepre-incubated in the presence of enzyme for 5 minutes prior to additionof ATP and the activity remaining quantified using the above describedactivity assay. The % Inhibition of a compound was determined using thefollowing formula; % Inhibition=100×(1−(experimental value−backgroundvalue)/(high activity control−background value)). The IC₅₀ value wasdetermined using a non-linear 4 point logistic curve fit (XLfit4, IDBS)with the formula; (A+(B/(1+((x/C){circumflex over ( )}D)))), whereA=background value, B=range, C=inflection point, D=curve fit parameter.

In Table 1 below, IC₅₀ value ranges for exemplary compounds are givenusing 100 uM ATP. The IC₅₀ ranges are indicated as “A,” “B,” and “C,”for values less than or equal to 0.1 μM; those greater than 0.1 μM andless than or equal to 0.5 μM; and those greater than 0.5 μM,respectively. IC₅₀ ranges denoted with an asterisk indicated that 16 μMATP (Sigma cat#A7699) was used in the assay.

EXAMPLE C Cancer Cell Line Data on Exemplary Compounds of the Invention

Breast cancer cells (MDA-MB-468), colon cancer cells (HCT116) andovarian cancer cells (OVCAR-3) were seeded (1000 to 4000 in 80 μl perwell depending on the cell growth rate) into 96 well plates 24 hoursbefore compound overlay. Compounds were prepared as 10 mM stocksolutions in 100% DMSO which were diluted with DMEM (Dulbecco's ModifiedEagle's Medium) cell growth Medium (Invitrogen, Burlington, ON, Canada)containing 10% FBS (Fetal Bovine Serum) to concentrations ranging from50 nM to 250 μM. Aliquots (20 μl) from each concentration were overlaidto 80 μl of the pre-seeded cells in the 96 well plates to make finalconcentrations of 10 nM to 50 μM. The cells were cultured for 5 daysbefore the Sulforhodamine B assay (SRB) was performed to determine thecompound's cell growth inhibition activity.

Sulforhodamine B (purchased from Sigma, Oakville, ON, Canada) is awater-soluble dye that binds to the basic amino acids of the cellularproteins. Thus, colorimetric measurement of the bound dye provides anestimate of the total protein mass that is related to the cell number.the cells are fixed in situ by gently aspirating off the culture mediaand adding 50 μl ice cold 10% Trichloroacetic Acid (TCA) per well andincubate at 4° C. for 30-60 mM, The plates are washed with H₂O fivetimes and allowed to air dry for 5 mM. Addition of 50 μl 0.4% (w/v) SRBsolution in 1% (v/v) acetic acid to each well and incubatation for 30 mMat RT completes the staining reaction. Following staining, plates arewashed four times with 1% acetic acid to remove unbound dye and thenallowed to air dry for 5 mM. The stain is solubilized with 100 μl of 10mM Tris pH 10.5 per well. Absorbance is read at 570 nm.

The percentage (%) of relative growth inhibition was calculated bycomparing to DMSO treated only cells (100%). GI₅₀'s were determined forcompounds with cytotoxic activity. The GI₅₀ was calculated usingGraphPad PRISM software (GraphPad Software, Inc., San Diego, Calif.,USA). GI₅₀ (growth inhibition) is the compound concentration that causes50% inhibition of cell growth.

In Table 1 below, GI₅₀ value ranges for compound examples against breastcancer cell lines (MDA-MB-468), colon cancer cell lines (HCT116) andovarian cancer cell lines (OVCAR-3) are given. The example compoundsdemonstrated varying growth inhibition/cell killing activity againstcells of breast cancer, colon cancer, and ovarian cancer. The GI₅₀ranges are indicated as “A,” “B,” and “C,” for values less than or equalto 0.1 μM; those greater than 0.1 μM and less than or equal to 0.5 μM;and those greater than 0.5 μM, respectively.

EXAMPLE D Colon and Ovarian Cancer Tumor-Initiating Cell Data ofExemplary Compounds

Materials and Methods: Non-tissue or tissure cultured treated T-75 flaskand 96-well plates were purchased from VWR. Vitamin B-27 supplement, MEMNEAA (minimum essential medium non-essential amino acids), sodiumpyruvate, L-glutamine, N2 supplement, penicillin-streptomycin andfungizone/amphotericin B were obtained from Invitrogen. Lipid mixture,heparin and EGF were purchased from Sigma; bFGF from BD Biosciences.Tumor Initiating Cells (TICs) from colon were routinely maintained usingnon-tissue cultured treated T-75 flasks in DMEM:F12 medium containing0.2XB-27 supplement, 4 ug/ml heparin, 1×MEM NEAA, 1×sodium pyruvate, 1mM glutamine, 10 pg/ul bFGF, 20 pg/ul EGF, 1× N2 supplement, lipidmixture, penicillin-streptomycin and fungizone/amphotericin B. OvarianTICs were routinely maintained using tissue cultured treated T-75 flasksin DMEM:F12 medium containing 1XB-27 supplement, 4 ug/ml heparin, 20pg/ul bFGF, 20 pg/ul EGF and penicillin-streptomycin.

Assay Protocol: Compounds described herein were dissolved in DMSO andfurther diluted in cell culture medium for GI₅₀ determination. ColonTICs were trypsinized and seeded into non-tissue cultured treated96-well plates with 4,000 cells/well. After 24 h, compound was addedinto the cell culture at different concentrations, and the finalconcentration of DMSO was adjusted to 0.1%. Cells were then cultured at37° C. for 9 days. Ovarian TICs were trypsinized and seeded into tissuecultured treated 96-well plates with 1,000 cells/well. After 24 h,compound was added into the cell culture at different concentrations,and the final concentration of DMSO was adjusted to 0.1%. Cells werethen cultured at 37° C. for 6 days. Cell viability was assessed byAlamar Blue assay: 10 ul of Alamar Blue was added into each well. After4 hours incubation at 37° C., fluorescence was recorded at excitation544 and emission 590. GI₅₀ (Growth inhibition) was calculated usingGraphPad Prism 4.0 software. Cell growth inhibition data for compoundsdescribed herein is tabulated below.

In Table 1 below, GI₅₀ value ranges for compound examples against TICs(Colon 12 and Ovarian 2393A) are given. The GI₅₀ ranges are indicated as“A,” “B,” and “C,” for values less than or equal to 0.1 μM; thosegreater than 0.1 μM and less than or equal to 0.5 μM; and those greaterthan 0.5 μM, respectively.

TABLE 1 In vitro activity of Compound Examples Cancer Cell TumorInitiating TTK Line GI₅₀ Range Cell GI₅₀ Range Example IC₅₀ MDA- OvarianColon # Range MB-468 HCT116 OVCAR-3 2393A 12 A1 A A A A A A A2 A A A AND ND A3 A A A A ND ND A4 A A A A A ND A5 A A A B ND ND A6 A A A A A AA8 A B B C ND ND A11 A A A A ND ND A12 A A A A ND ND A13 A B A B ND NDND—not determined

1-2. (canceled)
 3. A compound, wherein the compound is represented bythe following structural formula:

or a pharmaceutically acceptable salt thereof.
 4. (canceled)
 5. Acompound, wherein the compound is represented by the followingstructural formula:

or a pharmaceutically acceptable salt thereof.
 6. A compound, whereinthe compound is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.
 7. A pharmaceuticalcomposition comprising a compound of claim 3 or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier ordiluent.
 8. A method for treating cancer, the method comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 3 or a pharmaceutically acceptable salt thereof. 9.The method of claim 8, wherein the cancer is pancreatic cancer, prostatecancer, lung cancer, melanoma, breast cancer, colon cancer, or ovariancancer.
 10. A pharmaceutical composition comprising a compound of claim5 or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier or diluent.
 11. A method for treating cancer, themethod comprising administering to a subject in need thereof aneffective amount of the compound of claim 5 or a pharmaceuticallyacceptable salt thereof.
 12. The method of claim 11, wherein the canceris pancreatic cancer, prostate cancer, lung cancer, melanoma, breastcancer, colon cancer, or ovarian cancer.
 13. A pharmaceuticalcomposition comprising a compound of claim 6 or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier ordiluent.
 14. A method for treating cancer, the method comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 6 or a pharmaceutically acceptable salt thereof. 15.The method of claim 14, wherein the cancer is pancreatic cancer,prostate cancer, lung cancer, melanoma, breast cancer, colon cancer, orovarian cancer.